The invention pertains to the field of recombinant DNA technology and concerns a method for the preparation of the thrombin inhibitor desulphatohirudin with the aid of genetically engineered eukaryotic cells, said genetically engineered eukaryotic cells, hybrid vectors carrying a gene for desulphatohirudin and methods for the preparation of said eukaryotic cells and said hybrid vectors.
Hirudin is an anticoagulant agent that occurs naturally in leeches (Hirudo medicinalis). Hirudin is not a single protein species but consists of at least three components designated hirudin variant 1 (HV1), hirudin variant 2 (HV2) (cf. European Patent Application No. 158,564) and xe2x80x9cdes-(Val)2-hirudinxe2x80x9d (cf. European Patent Application No. 158,986). The variants differ in structure from each other by a number of amino acids (especially, the N-terminal sequence of HV1 is Val-Val-Tyr, that of HV2 is Ile-Thr-Tyr and that of xe2x80x9cdes-(Val)2-hirudinxe2x80x9d is Thr-Tyr) but have an accumulation of hydrophobic amino acids at the N-terminus and of polar amino acids at the C-terminus, a tyrosine residue (Tyr63) present as sulphate monoester, three disulphide bridges and the anticoagulant activity in common.
Hirudin, with a Ki-value (complex dissociation constant) of 6xc3x9710xe2x88x9211M, is the strongest thrombin inhibitor known and is characterised by a specific affinity to thrombin. Other enzymes of the blood coagulation cascade are not inhibited by hirudin. In contrast to heparin which is the preferred anticoagulant in conventional anticoagulation therapy, hirudin exerts its inhibiting action directly on thrombin and, unlike the former, does not act through antithrombin III. The only pharmacologically detectable effect of purified hirudin is the inhibition of blood coagulation and the prophylaxis of thrombosis. No effect on heart rate, respiration, blood pressure, thrombocyte count, fibrinogen and haemoglobin could be observed when administered intravenously to dogs, even in high doses. In tests on rats, pigs and dogs, hirudin has proved effective in experimental thrombosis (induced either by stasis or by the injection of thrombin), in endotoxin shock, and also in DIC (disseminated intravascular coagulation). Whenever direct comparison tests have been carried out, hirudin has proved to be superior to heparin. Furthermore, hirudin has an extremely low toxicity, is non antigenic and shows an almost complete clearance via the kidneys in a biologically active form.
Although long known, hirudin has not as yet achieved the broad therapeutic use that might be expected on the basis of its excellent biological properties. Its extremely limited availability is a serious drawback which stands in the way of its widespread use in medicine. Up to now, hirudin preparations have been obtainable essentially from natural material(leech extracts), which is expensive and difficult to obtain, and employing time-consuming and costly isolation and purification processes [cf. P. Walsmann et al., Pharmazie 36, 653 (1981); European Patent Application No. 158,986]. In view of the relatively long sequence of 65 amino acids also the conventional peptide synthesis offers little hope of success for economic reasons. New methods must therefore be applied for the manufacture of adequate amounts of hirudin that render possible detailed clinical tests of its therapeutic potential and its broad therapeutic use in anticoagulation therapy.
Such methods are offered especially by recombinant DNA technology. By means of this technology it is possible to manufacture the most varied physiologically active polypeptides by culturing correspondingly genetically modified host organisms. In this context, it has to be mentioned that hirudin is presumably produced by leeches via a desulphatohirudin precursor which is post-translationally sulphated. It is expected that hosts other than leech lack the specific sulphate-transferring enzyme system and will therefore produce desulphatohirudins rather than hirudins. However, the biological activity is not injured by the absence of the sulphate group as is evidenced by the desulphatohirudin protein obtained by enzymatic removal of the sulphate group from the phenolic hydroxy group of the Tyr 63 residue of the corresponding hirudin protein (cf. European Patent Application No. 142,860).
In the recently published European Patent Application No. 158,564 the production of desulphatohirudin variants 1 and 2 and of analogues thereof by means of E. coli cells transformed with plasmids containing the structural genes for the respective desulphatohirudirsis disclosed. The anticoagulant activity measured in cell extracts of cultured E. coli cells and given in terms of anti-thrombin units (xe2x80x9cATUxe2x80x9d) amounts to 3,000-4,000 ATU/D600/l culture which corresponds to a concentration of 0.2 mg hirudin/OD/l (based upon an estimated specific activity of 15,000-20,000 ATU/mg of pure hirudin).
Presumably the poor yield in hirudin activity obtained from transformed E. coli cells is attributable to an accumulation of most of the hirudin protein in an inactive form in the cytoplasm due to incorrect folding of the molecule. The correct folding depends on the formation of three disulphide bridges within the hirudin molecule which are essential for enzyme activity(cf. P. Walsmann et al., supra). Other naturally secreted mammalian proteins, such as bovine growth hormone, human tissue plasminogen activator and human xcex3-interferon are likewise essentially inactive when produced and accumulated in the cytoplasm of microorganisms [cf. R. A. Smith et al., Science 229, 1219 (1985)]. It is apparent that the secretion pathway may favor disulphide bond formation since most proteins containing disulphide bridges are extracellular. There are other features that make secretion most desirable:
Secreted proteins are generally easier to detect and to purify than intracellularly accumulated products;
secretion of desired products into the medium avoids the necessity of breaking up the host organisms in order to recover the product;
some heterologous proteins may have a toxic effect on the host organism. When secreted they are less likely to interfere with normal cellular functions;
secreted proteins are less likely to be digested by proteolytic enzymes than intracellularly accumulated proteins which are attacked by these enzymes upon disintegration of the cells.
Most secreted proteins are encoded on the DNA as preproteins with a signal peptide as an appended amino terminal extension of the mature amino acid sequence. The signal peptide plays an essential role during cotranslational insertion of the protein into the membranes of the endoplasmatic reticulum (ER). A signal peptidase cleaves the signal peptide in an early event on the luminal side of the ER. Further transport to the outer cell membrane involves Golgi and secretory vesicles. The protein is either secreted into the periplasmic space (e.g. acid phosphatase, invertase) or into the culture medium (e.g. xcex1 factor, killer toxin).
Since all eukaryotes seem to share the mechanisms for the expression of genetic information and for sorting the expressed proteins, the expression of eukaryotic genes proceeds with greater efficiency in an eukaryotic host than in E. coli. Among the eukaryotic organisms yeast is the easiest to handle and to cultivate. A number of heterologous proteins have been expressed successfully in yeast. However, it has not been possible so far to define essential features of a proteinxe2x80x94except for an appended signal peptidexe2x80x94that would allow efficient secretion into the medium. Therefore, it is not possible to make reliable predictions whether or not a protein will be secreted. Thus, while 90% of total glucoamylase produced by transformed yeast (containing the genetic information for pre-glucoamylase) is secreted into the medium (PCT-Patent Application No. 84/2921) and high titers of epidermal growth factor (EGF) are found in the medium of cultured yeast containing the EGF gene with the appended xcex1 factor signal peptide (European Patent Application No. 116,201),only minor amounts or traces of xcex2-endorphin (xcex1-factor signal peptide, PCT-Patent Application No. 84/4330), leukocyte interferon A (invertase signal peptide, European Patent Application No. 127,304), human xcex3-interferon, human serum albumin, bovine interferons xcex11 and xcex12, tissue plasminogen activator, rennin and human insulin-like growth factor (xcex1-factor signal peptide, European Patent Application No. 123,544), leukocyte interferons D and A, xcex3-interferon and human growth hormone (MGH) (interferon and MGH signal peptides, respectively, European Patent Application No. 88,632) are detectable in the medium, and no secretion of Pseudomonas carboxypeptidase G2(CPG2) [G2(CPG2) signal peptide, European Patent Application No. 121,352] and tissue plasminogen activator (PHO5 signal peptide, European Patent Application No. 143,081) by transformed yeast into the medium is observed.
Accordingly, whether or not a given protein having an appended signal peptide is secreted by yeast is unpredictable and depends above all on the selected protein.
Hence, it was uncertain, whether a selected protein, such as hirudin, having an appended signal peptide would be secreted, at least to a certain degree, by transformed host organisms, such as yeast.
Surprisingly, it has now been found that proteins with hirudin activity are secreted into the medium by eukaryotic host organisms which contain a DNA comprising a DNA sequence encoding a signal peptide upstream of and in reading frame with the structural gene for desulphatohirudin.
It is an object of the present invention to provide a method for the production and secretion of proteins with hirudin activity in an eukaryotic host organism. It is a further object of the invention to provide hybrid vectors comprising a DNA sequence encoding a signal peptide upstream of and in reading frame with the structural gene for desulphatohirudin, and eukaryotic host organisms transformed with said hybrid vectors.
1. Process for the production of desulphatohirudin
The invention concerns a method for the production of proteins with hirudin activity comprising culturing under appropriate nutrient conditions an eukaryotic host organism transformed with a hybrid vector having one or multiple DNA inserts each comprising an eukaryotic expression control sequence, a DNA segment consisting of a first DNA sequence encoding a signal peptide upstream of and in reading frame with a second DNA sequence coding for mature desulphatohirudin which DNA segment is under transcriptional control of said expression control sequence, and a DNA sequence containing eukaryotic transcription termination signals, and isolating the proteins with hirudin activity from the culture medium and, if desired, isolating optionally additional protein with hirudin activity which is cell associated from the cell interior, and, if desired, converting a resulting protein with hirudin activity into a different protein with hirudin activity.
The term xe2x80x9cDNA sequence coding for mature desulphatohirudinxe2x80x9d is intended to embrace all structural genes coding for mature proteins having hirudin activity which are known to exist in and/or can be isolated from the leech genome, such as the structural genes for mature desulphatohirudin variants HV1, HV2, PA and for des-(Val)2-desulphatohirudin (unless the latter is a mere artifact of expression of the HV1 gene). The term xe2x80x9cmature desulphatohirudinxe2x80x9d refers to desulphatohirudins which are devoid of any pre- and pro-sequences.
There are to be understood as xe2x80x9cproteins with hirudin activityxe2x80x9d those proteins obtained from the secreting host cells that have a thrombin-inhibiting action and a positive reaction with anti-hirudin antibodies and that have the primary structure of a desulphatohirudin, as well as modified, such as sulphated, derivatives thereof, e.g. the corresponding hirudins, and shortened derivatives thereof, such as desulphatohirudins lacking one to seven amino acids at the C-terminus.
Such proteins are especially those having the formulae 
in which X represents the dipeptide residue Val-Val-(HV1) or Thr (Des-(Val)2-hirudin) and R represents the phenolic hydroxy group of Tyr or a group of the formula xe2x80x94Oxe2x80x94SO3H, and the derivatives lacking the C-terminal amino acid Gln, the C-terminal dipeptide -Leu-Gln, the C-terminal tripeptide -Tyr(R)-Leu-Gln or the C-terminal tetrapeptide
-Glu-Tyr(R)-Leu-Gln, or 
(HV2) in which R has the meanings given aboveor another hirudin variant named hirudin PA which is produced by leech (J. Dodt, Thesis, University of Munich, FRG, 1984) and has the formula 
(PA) in which R has the meanings given above.
The preferred protein with hirudin activity is that of formula I in which X represents the residue Val-Val- and R represents the phenolic hydroxy group of Tyr, and the derivatives thereof that are shortened at the C-terminus by one to seven, especially one to four amino acids.
Suitable eukaryotic host organisms are cells of higher organisms, especially mammalians, in particular established human or animal cell lines, such as VERO or HeLa cells, or the Chinese hamster ovary (CHO) cell line, and yeast, such as Saccharomyces cerevisiae. The preferred host organism according to the present invention is yeast, especially strains of Saccharomyces cerevisiae. 
The eukaryotic host organism, especially yeast, containing the above DNA sequences is cultured using methods known in the art.
Thus, transformed yeast strains according to the invention are cultured in a liquid medium containing assimilable sources of carbon, nitrogen and inorganic salts.
Various carbon sources are usable. Examples of preferred carbon sources are assimilable carbohydrates, such as glucose, maltose, mannitol or lactose, or an acetate such as sodium acetate, which can be used either alone or in suitable mixtures. Suitable nitrogen sources include, for example, amino acids, such as casamino acids, peptides and proteins and their degradation products, such as tryptone, peptone or meat extracts, furthermore yeast extract, malt extract, corn steep liquor, as well as ammonium salts, such as ammonium chloride, sulphate or nitrate which can be used either alone or in suitable mixtures. Inorganic salts which may be used include, for example, sulphates, chlorides, phosphates and carbonates of sodium, potassium, magnesium and calcium. Additionally, the nutrient medium may also contain growth promoting substances. Substances which promote growth include, for example, trace elements, such as iron, zinc, manganese and the like, or individual amino acids.
To a varying extent, yeast cells transformed with autonomously replicating plasmids, for example, plasmids containing yeast 2xcexc plasmid DNA (infra) tend to lose the introduced hybrid plasmid. For this reason, such yeast cells have to be grown under selective conditions, i.e. conditions which require the expression of a plasmid-encoded gene for growth. Most selective markers currently in use and present in the hybrid vectors according to the invention (infra) are genes coding for enzymes of amino acid or purine biosynthesis. This makes it necessary to use synthetic minimal media deficient in the corresponding amino acid or purine base. However, genes conferring resistance to an appropriate biocide may be used as well [e.g. genes conferring resistance to cycloheximide, to the amino-glycoside G 418, to a heavy metal, or the like]. Yeast cells transformed with vectors containing antibiotic resistance genes are grown in complex media containing the corresponding antibiotic whereby faster growth rates and higher cell densities are reached.
Yeast cells transformed with DNA integrating into a chromosome do not require selective growth conditions. These transformed cells are sufficiently stable to allow growth without selective pressure. These cells are advantageously grown in complex media.
Yeast cells containing hybrid plasmids with a constitutive promoter (e.g. ADHI, GAPDH) express the desulphatohirudin gene controlled by said promoter without induction. However, if the desulphatohirudin gene is under the control of a regulated promoter (e.g. PGK or PHO5) the composition of the growth medium has to be adapted in order to obtain maximum levels of mRNA transcripts, i.e. when using the PHO5 promoter the growth medium must contain a low concentration of inorganic phosphate for derepression of this promoter.
The cultivation is carried out by employing conventional techniques. The culturing conditions, such as temperature, pH of the medium and fermentation time are selected in such a way that maximal levels of desulphatohirudin are produced. A chosen yeast strain is preferably grown under aerobic conditions in submerged culture with shaking or stirring at a temperature of about 25xc2x0 to 35xc2x0 C., preferably at about 30xc2x0 C., at a pH value of from 4 to 8, for example at approximately pH 7, and for about 4 to 20 hours, preferably until maximum yields of proteins are reached.
It is surprisingly found that, irrespective of the host organism and the signal peptide used, most of the produced hirudin compounds are secreted into the culture medium whereas only a minor part remains cell associated. The precise ratio (secreted compounds/cell associated compounds) depends on the fermentation conditions and the recovery process applied. In general it amounts to about or more than 9:1. Accordingly, secreted hirudin is always strongly dominating.
The hirudin compounds can be isolated from the culture medium by conventional means. For example, the first step consists usually in separating the cells from the culture fluid by means of centrifugation. The resulting supernatant can be enriched for hirudin compounds by treatment with polyethyleneimine so as to remove most of the non-proteinaceous material, and precipitation of the proteins by saturating the solution with ammonium sulphate or by trichloroacetic acid. Host proteins, if present, can also be precipitated by means of acidification with acetic acid (for example 0.1%, pH 4-5). A further enrichment of hirudin compounds can be achieved by extracting the acetic acid supernatant with n-butanol. Other purification steps include, for example, desalination, chromatographic processs, such as ion exchange chromatography, gel filtration chromatography, partition chromatography, HPLC, reversed phase HPLC and the like. The separation of the constituents of the mixture is also effected by dialysis, according to charge by means of gel electrophoresis or carrier-free electrophoresis, according to molecular size by means of a suitable Sephadex column, by affinity chromatography, for example with antibodies, especially monoclonal antibodies, or with thrombin coupled to a suitable carrier for affinity chromatography, or by other processes, especially those known from the literature.
If it is desired to isolate any additional hirudin compounds which are cell associated, i.e. which have accumulated intracellularly or in the periplasmic space, some supplementary purification steps are required. Thus, in case the hirudin proteins have accumulated within the cells, the first step for the recovery of the desired proteins consists in liberating the proteins from the cell interior. In most procedures the cell wall is first removed by enzymatic digestion with glucosidases (infra). Subsequently, the resulting speroplasts are treated with detergents, such as Triton. Alternatively, mechanical forces, such as shearing forces (for example X-press, French-press) or shaking with glass beads, are suitable for breaking cells. In the case where the hirudin compounds are secreted by the host, especially yeast, cells into the periplasmic space, a simplified protocol can be used: The protein is recovered without cell lysis by enzymatic removal of the cell wall or by treatment with chemical agents, e.g. thiol reagents or EDTA, which give rise to cell wall damages permitting the produced protein to be released.
It is surprisingly found that apart from the desulphatohirudin compounds corresponding to the xe2x80x9cDNA sequence coding for mature desulphatohirudinxe2x80x9d some other desulphatohirudin compounds can be isolated from the culture broth which differ from the expected compounds by the absence of one to four amino acids at the C-terminus. Thus, culturing of yeast cells carrying the gene coding for desulphatohirudin variant HV 1 yields this variant as well as, in lower yields, desulphatohirudin compounds lacking the C-terminal amino acid Gin [Des-(Gln65)-desulphatohirudin], the C-terminal dipeptide residue -Leu-Gln [Des-(Leu64, Gln65)-desulphatohirudin], the C-terminal tripeptide residue -Tyr-Leu-Gln [Des-(Tyr63, Leu64, Gln65)-desulphatohirudin] and the C-terminal tetrapeptide residue -Glu-Tyr-Leu-Gln [Des-(Glu62, Tyr63, Leu64, Gln65-desulphatohirudin], respectively. These compounds may have been formed by (partial) proteolytical degradation of the primary expression product desulphatohirudin and have been identified in all culture broths, irrespective of the yeast host used and the fermentation conditions applied.
Des-(Gln65)-desulphatohirudin, Des-(Tyr63, Leu64, Gln65)-desulphatohirudin and Des-(Glu62, Tyr63, Leu64, Gln65)-desulphatohirudin are novel and are also objects of the present invention.
The test with anti-hirudin or anti-desulphatohirudin antibodies (for example, monoclonal antibodies obtainable from hydridoma cells), the thrombin test [M. U. Bergmeyer (ed.), Methods in Enzymatic Analysis, Vol. II, p. 314-316, Verlag Chemie,Weinheim (FRG) 1983] or the blood coagulation test [F. Markwardt et al., Thromb. Haemost. 47, 226 (1982)] can be used to detect the hirudin activity.
Hirudin compounds obtainable in accordance with the process can be converted in a manner known per se into different hirudin compounds.
Thus, for example, compounds of the formula I to III in which R represents the phenolic hydroxy group of Tyr can be converted by reaction with a sulphate, such as disodium sulphate, and a tyrosine sulphotfansferase, obtainables for example, from leech cells, into compounds of the formula I to III in which R represents a group of the formula xe2x80x94OSO3H.
It is also possible to convert a desulphatohirudin compound obtained, for example desulphatohirudin variant HV 1, into a derivative thereof that lacks one to seven amino acids at the C-terminus, for example by the action of a suitable carboxypeptidase, such as carboxypeptidase Y.
The transformed eukaryotic host organisms according to the invention can be prepared by recombinant DNA techniques comprising the steps of
preparing a DNA construct containing an eukaryotic expression control sequence, a DNA segment consisting of a first DNA sequence encoding a signal peptide upstream of and in reading frame with a second DNA sequence coding for mature desulphatohirudin which DNA segment is under transcriptional control of said expression control sequence, and a DNA sequence containing eukaryotic transcription termination signals,
preparing a hybrid vector comprising said DNA construct,
transforming a suitable eukaryotic host organism with the created hybrid vector,
and selecting transformed host cells from untransformed host cells.
2. DNA constructs comprising the coding regions of a signal peptide and of desulphatohirudin
The invention concerns a DNA construct containing an eukaryotic expression control sequence, a DNA segment consisting of a first DNA sequence encoding a signal peptide upstream of and in reading frame with a second DNA sequence coding for mature desulphatohirudin which DNA segment is under transcriptional control of said expression control sequence, and a DNA sequence containing eukaryotic transcription termination signals.
Several expression control sequences can be used for regulator of expression of the DNA sequences coding for the signal peptide and desulfatohirudin. In particular, expression control sequences of highly expressed genes of the host to be transformed are used.
For use in mammalian cells, the expression control sequences are preferably derived from vira. For example, suitable expression control sequences for use in mammalian cells are the early and late promoters of Simian Virus 40 (SV40), the vaccinia promoter, HTLV promoter or promoters derived from polyoma or Adenovirus 2.
Expression control sequences for yeast which are the most preferred host organisms according to the present invention are derived from the genomic DNA of yeast, especially of Saccharomyces cerevisiae. Preferably, the expression control sequence of a highly expressed yeast gene is used for the expression of desulphatohirudin. Thus the promoter of the TRP1 gene, the ADHI or ADHII gene, acid phosphatase (PHO5) gene, isocytochrome c gene, or a promoter of the enolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 3-phosphoglycerate kinase (PGK), hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase and glucokinase genes, or a promoter of the yeast mating pheromone genes coding for the a- or xcex1-factor, can be used. It is also possible to use hybrid promoters comprising upstream activation sequences (UAS) of one yeast gene and downstream promoter elements including a functional TATA box of another yeast gene, for example a hybrid promoter including the UAS(s) of the yeast PHO5 gene and downstream promoter elements including a functional TATA box of the yeast GAPDH gene. Preferred vectors of the present invention contain promoters with transcriptional control. Promoters of this type, e.g. the promoter of the PHO5 gene and PHO5-GAPDH hybrid promoters, can be turned on or off by variation of the growth conditions. For example, the PHO5 promoter can be repressed or derepressed at will, solely by increasing or decreasing the concentration of inorganic phosphate in the medium. A further preferred promoter according to the invention is the promoter of the GAPDH gene, especially a functional fragment thereof starting at nucleotide xe2x88x92300 to xe2x88x92180, in particular at nucleotide xe2x88x92263 or xe2x88x92199, and ending at nucleotide xe2x88x925 of the GAPDH gene.
The DNA sequence encoding a signal peptide (xe2x80x9csignal sequencexe2x80x9d) is preferably derived from eukaryotic, for example yeast, genes coding for polypeptides which are ordinarily secreted. Suitable signal sequences are, for example, the hirudin signal sequence obtainable from genomic leech DNA, yeast signal sequences, such as the signalandprepro sequences of the yeast invertase, a-factor, xcex1-factor, pheromone peptidase, xe2x80x9ckiller toxinxe2x80x9d and repressible acid phospatase (PHO5)genes and the glucomylase signal sequence from Aspergillus awamori. Alternatively, fused signal sequences may be constructed by ligating part of the signal sequence (if present) of the gene naturally linked to the promoter used, with part of the hirudin signal sequence. Those combinations are favoured which allow a precise cleavage between the signal sequence and the mature desulphatohirudin amino acid sequence. Additional sequences, such as pro- or spacer-sequences which may or may not carry specific processing signals can also be included in the constructions to facilitate accurate processing of precursor molecules. Alternatively fused proteins can be generated containing internal processing signals which allow proper maturation in vivo or in vitro. For example, the processing signals contain a Lys-Arg residue, which is recognized by a yeast endopeptidase located in the Golgi membranes. The preferred signal sequences according to the present invention are those of the yeast PHO5 gene coding for a signal peptide having the formula
Met Phe Lys Ser Val Val Tyr Ser Ile Leu Ala Ala Ser Leu Ala Asn Ala,
and of the yeast invertase gene coding for a signal peptide having the formula
Met Leu Leu Gln Ala Phe Leu Phe Leu Leu Ala Gly Phe Ala Ala Lys Ile Ser Ala
The DNA sequence coding for mature desulphatohirudin is especially selected from the structural genes for desulphatohirudins specified above. The preferred DNA sequence codes for desulphatohirudin variant 1 (HV1) and has the formula
GTTGTTTACACCGACTGCACCGAATCTGGTCAGAACCTGTGCCTGTGCGAAGGT CAACAAATGTGGCTGACGTGGCTTAGACCAGTCTTGGACACGGACACGCTTCCA
TCTAACGTTTGCGGTCAGGGTAACAAATGCATCCTGGGTTCTGACGGTGAAAAAAACCAG AGATTGCAAACGCCAGTCCCATTGTTTACGTAGGACCCAAGACTGCCACTTTTTTTGGTC
TGCGTTACCGGTGAAGGTACCCCGAAACCGCAGTCTCACAACGACGGTGACTTCGAA ACGCAATGGCCACTTCCATGGGGCTTTGGCGTCAGAGTGTTGCTGCCACTGAAGCTT
GAAATCCCGGAAGAATACCTGCAG CTTTAGGGCCTTCTTATGGACGTC.
A DNA sequence containing eukaryotic transcription termination signals is preferably the 3xe2x80x2flanking sequence of an eukaryotic gene which contains proper signals for transcription termination and polyadenylation. Suitable 3xe2x80x2 flanking sequences are for example those of the gene naturally linked to the expression control sequence used. In case yeast is used as the host organism the preferred flanking sequences are those of the yeast PHO5 gene. Preferably, the DNA construct according to the present invention is provided at both ends with synthetic deoxynucleotide linkers which allow insertion and cloning of the construct in a cloning vector.
The eukaryotic expression control sequence, the DNA sequence coding for the signal peptide, the DNA sequence coding for mature desulphatohirudin and the eukaryotic transcription termination signals are operably linked to each other, i.e. they are juxtaposed in such a manner that their normal functions are maintained. Thus, the array is such that the expression control sequence effects proper expression of the signal peptide-desulphatohirudin gene complex, the transcription termination signals effect proper termination of transcription and polyadenylation and the signal sequence is linked to the desulphatohirudin gene in such a manner that secretion of desulphatohirudin occurs. Accordingly, if the expression control sequence and the signal sequence are derived from different genes, the expression control sequence is preferably joined to the signal sequence between the major mRNA start and the ATG of the gene naturally linked to the expression control sequence. The signal sequence should have its own ATG for translation initiation. The junction of these sequences is preferable effected by means of synthetic oligodeoxynucleotide linkers which may carry the recognition sequence of an endonuclease. On the other hand, the last codon of the signal sequence is directly linked to the first codon of the gene for desulphatohirudin.
The DNA constructs according to the invention may be prepared by methods known in the art, for example, by linking an eukaryotic expression control sequence, a DNA segment consisting of a first DNA sequence encoding a signal peptide upstream of and in reading frame with a second DNA sequence coding for desulphatohirudin, and a DNA sequence containing eukaryotic transcription termination signals in such a way that proper expression of the DNA segment and secretion of the produced desulphatohirudin is effected in an eukaryotic host organism.
Linkage of the various DNA sequences to yield the DNA constructs according to the invention may be effected by blunt end ligation or via suitable common restriction sites or synthetic linker molecules taking special care to a correct junction so that the normal functions of these DNA sequences are maintained. Thus, the signal sequence and the desulphatohirudin gene can be fused by blunt end ligation. Another approach for creating the correct junction of the signal sequence and the desulphatohirudin gene consists in restricting, if possible, the signal sequence near its 3xe2x80x2 terminus and the desulphatohirudin near its 5xe2x80x2 terminus so that each sequence lacks a predetermined number of base pairs. A synthetic oligodeoxynucleotide linker can then be constructed in such a way that, when joining the restricted signal sequence and the restricted desulphatohirudin gene via the connecting oligodeoxynucleotide, the missing base pairs are restored and the desulphatohirudin gene is in the proper reading frame relative to the signal sequence.
The DNA sequence coding for desulphatohirudin can be isolated from genomic leech DNA or a complementary double-stranded desulphatohirudin DNA (desulphatohirudin ds cDNA) is produced from desulphatohirudin mRNA, or a gene coding for the amino acid sequence of desulphatohirudin is produced by means of chemical and enzymatic processes. Genomic desulphatohirudin DNA and desulphatohirudin ds cDNA are obtained, for example, according to methods that are known per se. For example, genomic desulphatohirudin DNA is obtained from a leech gene bank that contains the desulphatohirudin gene by cloning the leech DNA fragments in a microorganism and identifying clones that contain desulphatohirudin DNA, for example by colony hybridisation using a radioactively labelled desulphatohirudin DNA-specific oligodeoxynucleotide that contains at least 15, and preferably from 15 to 30, deoxynucleotides. The resulting DNA fragments as a rule contain in addition to the desulphatohirudin gene other undesired DNA constituents that can be removed by treatment with suitable exo- or endo-nucleases.
Double-stranded desuiphatohirudin cDNA can be produced, for example, by isolating mRNA from suitable leech cells, which are preferably induced to form hirudin, enriching the desulphatohirudin mRNA in the resulting mRNA mixture in a manner known per se, using this mRNA as a template for the preparation of single-stranded cDNA, synthesising from this, with the aid of an RNA-dependent DNA polymerase, ds cDNA, and cloning the latter into a suitable vector. Clones that contain desulphatohirudin cDNA are identified, for example in the manner described above, by colony hybridisation using a radioactively labelled, desulphatohirudin DNA-specific oligodeoxynucleotide.
The desulphatohirudin gene can also be produced by chemical synthesis. The process is characterised in that segments of the coding and of the complementary strand of said gene are chemically synthesised and resulting segments are converted enzymatically into a structural gene for desulphatohirudin.
The chemical synthesis of DNA is well-known in the art and makes use of conventional techniques. Appropriate techniques have been compiled by S. A. Narang [Tetrahedron 39, 3 (1983)]. In particular, the methods described in European Patent Application No. 146,785 may be used and are herein incorporated by reference.
In an analogous manner, the signal sequence can be prepared by chemical synthesis or is isolated from chromosomal DNA of a suitable eukaryotic organism.
3. Hybrid Vectors Containing DNA Constructs with the Coding Regions of a Signal Peptide and of Desulphatohirudin
According to the present invention there is further provided a hybrid vector having one or multiple DNA inserts each comprising an eukaryotic expression control sequence, a DNA segment consisting of a first DNA sequence encoding a signal peptide upstream of and in reading frame with a second DNA sequence coding for mature desulphatohirudin which DNA segment is under transcriptional control of said expression control sequence, and a DNA sequence containing eukaryotic transcription termination signals.
The hybrid vectors according to the invention are selected from the group consisting of a hybrid plasmid and a linear DNA vector and are further selected depending on the host organism envisaged for transformation.
The invention relates especially to a linear DNA vector having one or multiple DNA inserts each comprising an eukaryotic expression control sequence, a DNA segment consisting of a first DNA sequence encoding a signal peptide upstream of and in reading frame with a second DNA sequence coding for mature desulphatohirudin which DNA segment is under transcriptional control of said expression control sequence, and a DNA sequence containing eukaryotic transcription termination signals, said expression control sequence and said sequence containing transcription termination signals being derived from the same eukaryotic gene. Especially, the invention relates to a linear DNA vector comprising the yeast PHO5 promoter, the above DNA segment and the 3xe2x80x2 flanking region of PHO5.
By virtue of the homologous 3xe2x80x2 and 5xe2x80x2 flanking sequences the whole linear DNA vector including the coding regions for the signal peptide and desulphatohirudin is stably integrated into the respective host chromosome, i.e. in the case of the yeast PHO5 promoter and 3xe2x80x2 flanking sequences of the yeast PHO5 gene at the PHO5 locus in yeast chromosome II.
The invention relates also especially to hybrid plasmids which apart from the expression control sequence, the above DNA segment and the sequence containing transcription termination signals contain additional DNA sequences which are inessential or less important for the function of the promoter, i.e. for the expression of the desulphatohirudin gene,but which perform important functions, for example, in the propagation of the cells transformed with said hybrid plasmids. The additional DNA sequences may be derived from prokaryotic and/or eukaryotic cells and may include chromosomal and/or extra-chromosomal DNA sequences. For example, the additional DNA sequences may stem from (or consist of) plasmid DNA, such as bacterial or eukaryotic plasmid DNA, viral DNA and/or chromosomal DNA, such as bacterial, yeast or higher eukaryotic chromosomal DNA. Preferred hybrid plasmids contain additional DNA sequences derived from bacterial plasmids, especially Escherichia coli plasmid pBR322 or related plasmids, bacteriophage xcex, yeast 2xcexc plasmid, and/or yeast chromosomal DNA.
In the preferred hybrid plasmids according to the invention, the additional DNA sequences carry a yeast replication origin and a selective genetic marker for yeast. Hybrid plasmids containing a yeast replication origin, e.g. an autonomously replicating segment(ars), are extrachromosomally maintained within the yeast cells after transformation and are autonomously replicated upon mitosis. Hybrid plasmids containing sequences homogous to yeast 2xcexc plasmid DNA can be used as well. These hybrid plasmids will get integrated by recombination into 2xcexc plasmids already present within the cell or will replicate autonomously.
As to the selective gene marker for yeast, any marker gene can be used which facilitates the selection for transformants due to the phenotypic expression of the marker. Suitable markers for yeast are particularly those expressing antibiotic resistance or, in the case of auxotrophic yeast mutants, genes which complement host lesions. Corresponding genes confer, for example, resistance to the antibiotic cycloheximide or provide for prototrophy in an auxotrophic yeast mutant, for example the URA1, URA3, ARG4, LEU2, HIS4, HIS3, TRP5 or TRP1 gene.
Advantageously, the additional DNA sequences which are present in the hybrid plasmids according to the invention also include a replication origin and a selective genetic marker for a bacterial host, especially Escherichia coli. There are useful features which are associated with the presence of an E. coli replication origin and an E. coli marker in a yeast hybrid plasmid. Firstly, large amounts of hybrid plasmid DNA can be obtained by growth and amplification in E. coli and, secondly, the construction of hybrid plasmids is conveniently done in E. coli making use of the whole repertoire of cloning technology based on E. coli. E. coli plasmids, such as pBR322 and the like, contain both E. coli replication origin and E. coli genetic markers conferring resistance to antibiotics, for example tetracycline and ampicillin, and are advantageously employed as part of the yeast hybrid vectors.
The additional DNA sequences which contain, for example, replication origin and genetic markers for yeast and a bacterial host (see above) are hereinafter referred to as xe2x80x9cvector DNAxe2x80x9d which together with the above DNA construct, containing inter alia the expression control sequence and the desulphatohirudin gene, is forming a hybrid plasmid according to the invention.
The hybrid vectors according to the invention may contain one or multiple DNA inserts each comprising, inter alia, the expression control sequence, the DNA sequence encoding the signal peptide and the DNA sequence coding for mature desulphatohirudin. If the hybrid vectors contain multiple DNA inserts, preferably 2 to 4 DNA inserts, these can be present in a tandem array or at different locations of the hybrid vector. Preferred hybrid vectors contain one DNA insert or DNA inserts in a tandem array. The DNA inserts are especially head to tail arranged.
The hybrid plasmids according to the invention are prepared by methods known in the art. The process for the preparation of the hybrid vectors comprises introducing one or multiple DNA constructs containing an eukaryotic expression control sequence, a DNA segment consisting of a first DNA sequence encoding a signal peptide upstream of and in reading frame with a second DNA sequence coding for mature desulphatohirudin which DNA segment is under transcriptional control of said expression control sequence, and a DNA sequence containing eukaryotic transcription termination signals, as such or introducing the components of said DNA constructs successively in the predetermined order into a vector DNA.
The construction of the hybrid plasmids according to the invention is performed applying conventional ligation techniques. The components of the plasmids are linked through common restriction sites and/or by means of synthetic linker molecules and/or by blunt end ligation.
The linear DNA vectors according to the invention correspond essentially to the DNA constructs specified in paragraph 2 and are prepared in an analogous manner.
4. Transformed eukaryotic host organisms
Another aspect of the invention involves eukaryotic host organisms transformed with a hybrid vector having one or multiple DNA inserts each comprising an eukaryotic expression control sequence, a DNA segment consisting of a first DNA sequence encoding a signal peptide upstream of and in reading frame with a second DNA sequence coding for mature desulphatohirudin which DNA segment is under transcriptional control of said expression control sequence, and a DNA sequence containing eukaryotic transcription termination signals, and mutants thereof.
Suitable eukaryotic host organisms are especially those specified above, in particular yeast including species of the genera Kluyveromyces, Candida, Pichia, Yarrowia, Saccharomyces, Schizosaccharomyces, Torulopsis, and related genera (cf. J. Lodder, The Yeasts, Amsterdam 1971), especially strains of Saccharomyces cerevisiae. 
The transformed eukaryotic host organisms are selected from an eukaryotic host organism transformed with a hybrid plasmid having one or multiple DNA inserts each comprising an eukaryotic expression control sequence, a DNA segment consisting of a first DNA sequence encoding a signal peptide upstream of and in reading frame with a second DNA sequence coding for mature desulphatohirudin which DNA segment is under transcriptional control of said expression control sequence, and a DNA sequence containing eukaryotic transcription termination signals, and an eukaryotic host organism with said DNA insert(s) stably integrated in a host chromosome.
The process for the production of said transformed eukaryotic host cells comprises transforming eukaryotic host cells with a hybrid vector having one or multiple DNA inserts each comprising an eukaryotic expression control sequence, a DNA segment consisting of a first DNA sequence encoding a signal peptide upstream of and in reading frame with a second DNA sequence coding for mature desulphatohirudin which DNA segment is under transcriptional control of said expression control sequence, and a DNA sequence containing eukaryotic transcription termination signals.
The transformation of the eukaryotic host cells is accomplished by methods known in the art. For example, the transformation of yeast with the hybrid vectors may be accomplished according to the method described by Hinnen et al [Proc. Natl. Acad. Sci. USA 75, 1929 (1978)]. This method can be divided into three steps:
(1) Removal of the yeast cell wall or parts thereof using various preparations of glucosidases, such as snail gut juices (e.g. Glusulase(copyright) or Helicase(copyright)) or enzyme mixtures obtained from microorganisms (e.g. Zymolyase(copyright)) in osmotically stabilized solutions (e.g. 1M sorbitol).
(2) Treatment of the xe2x80x9cnakedxe2x80x9d yeast cells (spheroplasts) with the DNA vector in the presence of PEG (polyethyleneglycol) and Ca2+ ions.
(3) Regeneration of the cell wall and selection of the transformed cells in a solid layer of agar.
This regeneration is conveniently done by embedding the spheroplasts into agar. For example, molten agar (about 50xc2x0 C.) is mixed with the spheroplasts. Upon cooling the solution to yeast growth temperatures (about 30xc2x0 C.), a solid layer is obtained. This agar layer is to prevent rapid diffusion and loss of essential macromolecules from the spheroplasts and thereby facilitates regeneration of the cell wall. However, cell wall regeneration may also be obtained (although at lower efficiency) by plating the spheroplasts onto the surface of preformed agar layers.
Preferably, the regeneration agar is prepared in a way to allow regeneration and selection of transformed cells at the same time. Since yeast genes coding for enzymes of amino acid biosynthetic pathways are generally used as selective markers (supra), the generation is preferably performed in yeast minimal medium agar. If very high efficiencies of regeneration are required the following two step procedure is advantageous: (1) regeneration of the cell wall in a rich complex medium, and (2) selection of the transformed cells by replica plating the cell layer onto selective agar plates.
When the above linear DNA vector is used for transforming the eukaryotic host cells, transformation is preferably done in the presence of a second vector containing a selective marker for yeast. This cotransformation allows enrichment for those host cells which have taken up DNA that cannot be directly selected for. Since competent cells take up any type of DNA a high percentage of cells transformed with a selective vector will also harbor any additional DNA (such as the above linear DNA vector).
The transformed eukaryotic host organisms, especially the transformed yeast strains, containing the hybrid plasmids according to the invention or having the linear DNA vector comprising the desulphatohirudin gene stably integrated in a host chromosome can be improved in production of desulphatohirudin by mutation and selection using methods known in the art. The mutation can be effected, for example, by U.V. irradiation or suitable chemical reagents. Especially preferred is the production of protease deficient mutants, especially yeast mutants, so as to avoid proteolytic degradation of the produced desulphatohirudin within the cells. Suitable mutants can be selected and isolated by conventional means.
The invention relates especially to the DNA constructs comprising the desulphatohirudin gene, the hybrid vectors, the transformed eukaryotic host organisms and to the processes for the preparation thereof as well as to the process for the production of proteins with hirudin activity as described in the Examples.